Gold nanoparticle-enhanced X-ray microtomography of the rodent reveals region-specific cerebrospinal fluid circulation in the brain

Cerebrospinal fluid (CSF) is essential for the development and function of the central nervous system (CNS). However, the brain and its interstitium have largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with the CSF. Here, we develop a technique for CSF tracking, gold nanoparticle-enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF circulation patterns during development. Using this method and subsequent histological analysis in rodents, we identify previously uncharacterized CSF pathways from the subarachnoid space (particularly the basal cisterns) that mediate CSF-parenchymal interactions involving 24 functional-anatomic cell groupings in the brain and spinal cord. CSF distribution to these areas is largely restricted to early development and is altered in posthemorrhagic hydrocephalus. Our study also presents particle size-dependent CSF circulation patterns through the CNS including interaction between neurons and small CSF tracers, but not large CSF tracers. These findings have implications for understanding the biological basis of normal brain development and the pathogenesis of a broad range of disease states, including hydrocephalus.


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March 2021

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The following softwares were used to analyze the data in this study: -FIJI image processing software (NIH) version 2.3.0/1. No human research participants were included in this study.
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Statistical methods were not used to re-calculate or predetermine sample sizes in any experiments. Sample size was determined based on similar studies with XRM/MicroCT and is consistent with recently published studies utilizing XRM/MicroCT (Maes et al, Nature Communications, October 2022). Since this study introduced a novel use of XRM to imaging CSF pathways, we primarily aimed to show the applicability and repeatability of this technique to tracking CSF, for which we deemed n = 3 for each condition was sufficient for proof-of concept. Our findings with XRM were also confirmed with additional histological analyses.
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To ensure reproducibility, all XRM data presented in this manuscript were repeated three times. All fluorescent tracer injections, immunofluorescence, MRI and histological experiments were repeated at least three times. All confocal and histological images presented were imaged at least three times for a single data point. Results for all technical and biological replicates were consistent among their groups. The exact number of replications of each experiment is stated in the corresponding Figure Legend.
All samples were randomly allocated into experimental groups by randomly choosing pups from each litter to receive different treatments. Rats used in this study were randomly chosen from their litters to receive artificial CSF or hemoglobin injections to create the control and PHH conditions. Rats were also randomly chosen to receive 1.9 nm or 15 nm gold nanoparticle injection or Red Dextran injection. Rats were also Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Laboratory animals randomly chosen to receive intracisterna magna vs. intraventricular injection. Mice and zebrafish used in this study were also randomly chosen to receive tracer injections.
Blinding was not performed for MRI image acquisition because imaging occurred shortly following the artificial CSF or hemoglobin injection, so the same experimenter performed injection and MRI acquisition. Experimental conditions were also evident from the MRI image data.
Blinding was performed for all other parts of this study, including XRM image acquisition and post-processing, gold nanoparticle and other tracer injection, immunostaining, histology, tissue harvesting, all quantifications and analyses.
Sprague Dawley Rats (Charles River Laboratories, Wilmington, MA) and C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were housed in a 12-hour light-dark cycle in a temperature and humidity-controlled room. Water and food were provided ad libitum. Rat and mouse pups were obtained from Charles River/Jackson Laboratories with their dams and housed at the animal facility with

March 2021
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